Prion diseases, such as Creutzfeldt-Jakob Disease (CJD), are degenerative disorders affecting the central nervous system. They are uniformly fatal and illicit death following a long period of steady neurodegeneration, dementia and motor disfunction. In prion diseases, an aberrantly folded conformer ofthe prion protein (PrPSc) propagates by catalyzing the post-translational misfolding of cellular PrP (PrPC). We have recently succeeded in recapitulating this phenomenon in vitro by converting purified, bacterially-expressed, recombinant PrP into infectious prions. The ability to create synthetic prions in cell-free systems has provided definitive proof of the protein-only hypothesis of prion propagation and has enabled us to begin deciphering the structural basis of protein-based infectivity. The specific aims of this project seek to utilize synthetic prions to gain insights into the mechanism of prion-induced neurodegeneration. We propose to create homogeneous and highly infectious preparations of synthetic prions. The synthetic prion preparations will be propagated in mouse bioassays and used for structural analyses. Disease phenotypes induced by synthetic prions will be assessed by two novel approaches for global analysis of proteome homeostasis. Effects on protein translation will be studied by ribosome profiling and changes in proteome turnover will be measured by comprehensive in vivo isotopic replacement. We will also attempt to infect induced neuronal (iN] cells with synthetic prions. These strategies will uncover relationships between the physical structure of prion strains and their biological properties and may provide valuable insights into the mechanism of neurodegeneration in prion diseases such as CJD.